The overall goal of this proposal is to dissect the mechanisms of immune regulation of chronic experimental autoimmune encephalomyelitis (EAE). EAE is an inflammatory disease of the central nervous system initiated by myelin antigen-specific encephalitogenic CD4+ Th1 cells. During an immune response, the T cell requires 2 signals for full activation. The first signal is provided by engagement of the TCR with the antigenic peptide plus MHC molecule on antigen-presenting cells (APCs), and the second "costimulatory" signal is provided by binding of specific receptors on T cells with their ligand/s on APCs. Investigations in the past few years have focused on the CD28-B7 and the CD4O-CD 154 pathways as the major costimulatory pathways for T cell activation. However, it has become increasingly clear that other costimulatory molecules participate in this function and that other molecules providing signal 2 may have an important role in T cell effector and memory function and the type of cytokines produced. This proposal will focus on understanding the interplay between the encephalitogenic and regulatory functions of T helper cells and novel costimulatory pathways in chronic EAE. In Aim 1 we will investigate the mechanisms of cytokine regulation in EAE: Our hypothesis is that Th1 cells mediate acute and chronic EAE, while Th2 cytokines may regulate EAE although this may not be universal. Using STAT4 and STAT6 KO mice and Thl and Th2 lines we will dissect the encephalitogenic function of Th1 and the regulatory functions of Th2 cells and cytokines in chronic EAE. We will also investigate the role of CTLA4 regulation of Th1 and Th2 autoimmune responses. In Aim 2 we will investigate the role of novel costimulatory pathways in regulation of Th1 and Th2 responses. Using anti-CD3OL, anti-OX4OL, and anti-ICOSL we will study the role of each of these pathways in Th1 versus Th2 responses, in generating memory and effector cells, and in cell survival. In Aim 3, we will investigate the role of cytokines in modifying the immune response of a biased T cell repertoire. Our hypothesis is that even in the presence of a biased T cell repertoire such as in a myelin antigen TCR transgenic animal, the cytokine milieu has a major role in expression of the disease phenotype. We will use MOG TCR transgenic mice intercrossed with STAT4 and STAT6 KO mice to study the occurrence of active and adoptive transfer disease. We will also study the in vivo mechanisms of the novel costimulatory pathways by adoptive transfer of STAT4 KO/TCR transgenic or STAT4 KO/TCR transgenic cells into WT or TCRalphabeta KO recipients.